Heparin/heparosan synthase from P. multocida and methods of making and using same

ABSTRACT

The presently claimed and disclosed invention relates, in general, to dual action heparin synthases and, more particularly, to dual action heparin synthases obtained from  Pasteurella multocida . The presently claimed and disclosed invention also relates to heparosan, heparin and heparin-like molecules provided by recombinant techniques and methods of using such molecules and also the identification or prediction of heparin synthases or component single action enzymes. The presently claimed and disclosed invention also relates to methods, and molecules produced according to such methods, for using the presently claimed and disclosed heparosan and/or heparin synthase for polymer grafting and the production of non-naturally occurring chimeric polymers incorporating stretches of one or more acidic GAG molecules, such as heparin, chondroitin, hyaluronan, and/or heparosan.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) of U.S.Provisional Application Ser. No. 60/289,554, filed May 8, 2001, entitled“PASTEURELLA MULTOCIDA HEPARIN SYNTHASE GENE AND METHODS OF MAKING ANDUSING SAME;” U.S. Provisional Application Ser. No. 60/296,386, filedJun. 6, 2001, entitled “HEPARIN AND HEPARIN-LIKE POLYSACCHARIDES, THEIRSYNTHASES, AND USES THEREOF;” U.S. Provisional Application Ser. No.60/303,691, filed Jul. 6, 2001, entitled “ENABLEMENT OF RECOMBINANTHEPARIN SYNTHASE, pmHAS;” and U.S. Provisional Application Ser. No.60/313,258, filed Aug. 17, 2001, entitled “HEPARIN SYNTHASE SEQUENCEMOTIFS AND METHODS OF MAKING AND USING SAME;” the contents of which arehereby expressly incorporated in their entirety by reference.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

The government owns certain rights in and to this application pursuantto a grant from the National Science Foundation (NSF), Grant No.MCB-9876193.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The presently claimed and disclosed invention relates, in general, todual action heparin synthases and, more particularly, to dual actionheparin synthases obtained from Pasteurella multocida. The presentlyclaimed and disclosed invention also relates to heparosan, heparin andheparin-like molecules produced according to recombinant techniques andmethods of using such molecules. The presently claimed and disclosedinvention also relates to methods, and molecules produced according tosuch methods, for using the presently claimed and disclosed heparosanand/or heparin synthases for polymer grafting and the production ofnon-naturally occurring chimeric polymers incorporating stretches of oneor more acidic GAG molecules, such as heparin, chondroitin, hyaluronan,and/or heparosan.

2. Background Information Relating to this Application

Glycosaminoglycans [GAGs] are long linear polysaccharides consisting ofdisaccharide repeats that contain an amino sugar and are found in mostanimals. Chondroitin [β(1,4)GlcUA-β(1,3)GalNAc]_(n), heparin/heparosan[β1,4)GlcUA-[α(1,4)GlcNAc]_(n), and hyaluronan[β(1,4)GlcUA-β(1,3)GlcNAc]_(n) are the three most prevalent GAGs foundin humans and are also the only known acidic GAGs. Chondroitin andheparin typically have n=20 to 100, while hyaluronan typically hasn=10³. Chondroitin and heparin are synthesized as glycoproteins and aresulfated at various positions in vertebrates. Hyaluronan is not sulfatedin vertebrates. A substantial fraction of the GlcUA residues of heparinand chondroitin are epimerized to form iduronic acid. A simplifiednomenclature has been developed for these GAGs. For example,heparin/heparosan's structure is noted as β4-GlcUA-α4-GlcNAc.

The capsular polysaccharide produced by the Type D strain of Pasteurellamultocida is N-acetyl heparosan (heparosan is unmodified heparin—i.e.sulfation or epimerization have not occurred). In vertebrates, one ormore modifications including O-sulfation of certain hydroxyls,deacetylation and subsequent N-sulfation, or epimerization of glucuronicacid to iduronic acid modifies the precursor N-acetyl heparosan toheparin/heparan. Hereinafter, for convenience and/or ease of discussion,heparin and/or heparosan are defined as polymers having theβ4GlcUA-α4GlcNAc backbone.

Many lower animals possess these same GAGs or very similar molecules.GAGs play both structural and recognition roles on the cell surface andin the extracellular matrix. By virtue of their physicalcharacteristics, namely a high negative charge density and a multitudeof polar hydroxyl groups, GAGs help hydrate and expand tissues. Numerousproteins bind selectively to one or more of the GAGs. Thus the proteinsfound on cell surfaces or the associated extracellular matrices (e.g.CD44, collagen, fibronectin) of different cell types may adhere orinteract via a GAG intermediate. Also GAGs may sequester or bind certainproteins (e.g. growth or coagulation factors) to cell surfaces.

Certain pathogenic bacteria produce an extracellular polysaccharidecoating, called a capsule, which serves as a virulence factor. In a fewcases, the capsule is composed of GAG or GAG-like polymers. As themicrobial polysaccharide is identical or very similar to the host GAG,the antibody response is either very limited or non-existent. Thecapsule is thought to assist in the evasion of host defenses such asphagocytosis and complement. Examples of this clever strategy ofmolecular camouflage are the production of an authentic HApolysaccharide by Gram-negative Type A Pasteurella multocida andGram-positive Group A and C Streptococcus. The HA capsule of thesemicrobes increases virulence by 10² to 10³-fold as measured by LD₅₀values, the number of colony forming units that will kill 50% of thetest animals after bacterial challenge.

The invasiveness and pathogenicity of certain E. coli strains has alsobeen attributed to their polysaccharide capsules. Two Escherichia colicapsular types, K4 and K5, make polymers composed of GAG-like polymers.The E. coli K4 polymer is an unsulfated chondroitin backbone decoratedwith fructose side-branches on the C3 position of the GlcUA residues.The K5 capsular material is a polysaccharide, called heparosan,identical to mammalian heparin except that the bacterial polymer isunsulfated and there is no epimerization of GlcUA to iduronic acid.

The studies of GAG biosynthesis have been instrumental in understandingpolysaccharide production in general. The HA synthases were the firstGAG glycosyltransferases to be identified at the molecular level. Theseenzymes utilize UDP-sugar nucleotide substrates to produce largepolymers containing thousands of disaccharide repeats. The genesencoding bacterial, vertebrate, and viral HAS enzymes have been cloned.In all these cases, expression studies have demonstrated thattransformation with DNA encoding a single HAS polypeptide conferred theability of foreign hosts to synthesize HA. Except for the most recentHAS to be identified, P. multocida pmHAS, these proteins have similaramino acid sequences, repeating conserved amino acid motifs, andpredicted topology in the membrane. Likewise, as presently disclosed andclaimed herein, heparosan and/or heparin synthases have been identifiedthat confer upon a foreign host the ability to produce heparin.

With respect to related microbial GAG synthases other than the HASs, theE. coil K5 heparin glycosyltransferases, KfiA (SEQ ID NO:8) and KfiC(SEQ ID NO:9), have been identified by genetic and biochemical means.These K5 glycosyltransferases synthesize heparosan (unsulfated andunepimerased heparin) in vivo. The KfiA and KfiC require KfiB (SEQ IDNO:10), an accessory protein, with unknown function in order tosynthesize heparosan, however. In vitro, the reactions are limited toadding one or two sugars; as such, it appears that some co-factor orreaction condition is missing—thus, extended polymerization does notoccur in vitro when KfiA, KfiB, and KfiC are used. As such, thepresently claimed and disclosed heparosan/heparin synthases provide anovel heretofore unavailable means for recombinantly producing heparin(the sulfated and epimerized molecule). In contrast to the HASs, thepmCS chondroitin synthase(s), and the presently disclosed and claimedheparin synthases, it appears that K5 requires two proteins, KfiA andKfiC, to transfer the sugars of the disaccharide repeat to the growingpolymer chain. The presently claimed and disclosed heparin synthases(designated “pmHS and PglA”) are dual action enzymes capable oftransferring both sugars of the growing heparin polymer chain. Theseenzymes polymerize heparosan in vivo and in vitro.

Many P. multocida isolates produce GAG or GAG-like molecules as assessedby enzymatic degradation and removal of the capsule of living bacterialcells. Type A P. multocida, the major fowl cholera pathogen, makes acapsule that is sensitive to hyaluronidase. Subsequent NMR structuralstudies of capsular extracts confirmed that HA was the majorpolysaccharide present. A specific HA-binding protein, aggrecan, alsointeracts with HA from Type A P. multocida. Two other distinct P.multocida types, a swine pathogen, Type D, and a minor fowl cholerapathogen, Type F, produce polymers that are chondroitin orchondroitin-like based on the observation that their capsules aredegraded by Flavobacterium chondroitin AC lyase. After enzymatic removalof the capsule, both types were more readily phagocytosed by neutrophilsin vitro. The capsule of Type D cells, but not Type F cells, also appearto be degraded by heparinase III, indicating that a heparin-typemolecule is present as well.

Heparin acts as an anticoagulant and is used to avoid coagulationproblems during extra corporal circulation and surgery as well as fortreatment after thrombosis has been diagnosed. Heparin is used in theprevention and/or treatment of deep venous thrombosis, pulmonaryembolism, mural thrombus after myocardial infarction, post thrombolyticcoronary rethrombosis, unstable angina, and acute myocardial infarction.In addition to use as a treatment for various medical conditions,heparin is also used to coat medical instruments and implants, such asstents, to prevent blood clotting. Using heparin to coat various medicalitems eliminates the need to prescribe anti-clotting medication in somecases.

Where heparin is used to treat medical conditions as those describedabove, two different methods and two different types of heparin areused. The two methods are intravenous infusion of standard heparin andinjection of low molecular mass heparin. Patients undergoing intravenousinfusion are hospitalized and the activated partial thromoplastin time(aPTT) is monitored. This type of treatment requires that the patientremain hospitalized until warfarin is administered to achieve anInternational Normalized Ratio (INR) between 2.0 and 3.0 often resultingin a three to seven day hospital stay. The alternative treatmentinvolves twice daily injections of low-molecular-weight heparin. Theinjection treatment allows the patient to self-administer or have avisiting nurse or family member administer the injections.

Low molecular weight heparin has a molecular weight of 1,000 to 10,000Daltons as compared to the molecular weight of standard heparin of 5,000to 30,000 Daltons. Low molecular weight heparin binds less strongly toprotein than standard heparin, has enhanced bioavailability, interactsless with platelets and yields more predictable blood levels. Thepredictability of blood levels eliminates the need to monitor the aPPT.In addition, low molecular weight heparin offers a lower likelihood ofbleeding and no reports of thrombocytopenia or osteoporosis have beenissued with respect to low molecular weight heparin.

In the presently claimed and disclosed invention, the monosaccharidecomposition of the P. multocida Type D polysaccharide has beenidentified and analyzed. The DNA sequence information of the Type A HAbiosynthesis locus and the Type F biosynthesis locus allowed for theprediction of the general properties of the Type D locus. From thisinformation on potential precursor genes required by a heparin synthase,pmHS was identified (P. multocida Heparin Synthase), the first dualaction microbial heparin synthase to be identified and molecularlycloned from any source. With respect to the pmHS, a single polypeptideis responsible for the copolymerization of the GlcUA and GlcNAcsugars—i.e. it is a dual action enzyme as opposed to the single actionnature of the at least three enzymes of E. coli K5 heparosanbiosynthesis locus that are required for heparin production. Theidentification of pmHS also allowed for the identification of theexistence of another heparin synthase found in Type A, D and F P.multocida. A gene with unknown function, called PglA, was found in agenome sequencing project of Type A P. multocida; no enzymatic function(or any function) has been previously described with respect to thisPglA gene. Hereinafter, and is contemplated and included within thepresently disclosed and claimed invention, is disclosed that the PglAenzyme, which is 70% identical to pmHS, is also a heparin synthase. Thisunexpected cryptic gene is functional in vitro in recombinant systems.The Type D capsular polymer has been identified as a heparin polymer.Organisms with the heparin synthase gene (Type D P. multocida) as newsources of heparin polymer have also been identified, purified, andcharacterized.

SUMMARY OF THE INVENTION

The presently claimed and disclosed invention relates, in general, todual action heparin synthases and, more particularly, to dual actionheparin synthases obtained from Pasteurella multocida. The presentlyclaimed and disclosed invention also relates to heparosan, heparin andheparin-like molecules produced according to recombinant techniques andmethods of using such molecules. The presently claimed and disclosedinvention also relates to methods, and molecules produced according tosuch methods, for using the presently claimed and disclosed heparosanand/or heparin synthases for polymer grafting and the production ofnon-naturally occurring chimeric polymers incorporating stretches of oneor more acidic GAG molecules, such as heparin, chondroitin, hyaluronan,and/or heparosan.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 graphically depicts Sequence Similarity of pmHS with KfiA andKfiC. Elements of the Pasteurella heparosan synthase, HS1 (containingresidues 91-240; SEQ ID NO:23) and HS2 (containing residues 441-540; SEQID NO:26) are very similar to portions of two proteins from the E. coilK5 capsular locus (A, residues 75-172 of KfiA, SEQ ID NO:27; C, residues262-410 of KfiC, SEQ ID NO:24) as shown by this modified Multalinalignment (ref. 21; numbering scheme corresponds to the pmHS sequence).The HS1 and HS2 elements may be important for hexosamine transferase orfor glucuronic acid transferase activities, respectively. (con,consensus symbols: asterisks, [K or R]and [S or T]; %, any one of F,Y,W;$, any one of L,M; !, any one of I,V; #, any one of E,D,Q,N). Theconsensus sequence of the alignment of HS1 and KfiC has been assignedSEQ ID NO:25, while the consensus sequence of the alignment of HS2 andKfiA is assigned SEQ ID NO:28.

FIG. 2 depicts pmHS Activity Dependence on Acceptor and EnzymeConcentration. Various amounts of crude membranes containing thefull-length enzyme, pmHS1-617, were incubated in 50 μl of buffercontaining 50 mM Tris, pH 7.2, 10 mM MgCl₂, 10 mM MnCl₂, 500 μMUDP-[¹⁴C]GlcUA (0.15 μCi), and 500 μM UDP-GlcNAc. Three parallel sets ofreactions were performed with either no acceptor (circles) or twoconcentrations of heparosan polymer acceptor (uronic acid: 0.6 μg,squares; 1.7 μg, triangles). After 40 min, the reaction was terminatedand analyzed by paper chromatography. The background incorporation dueto vector membranes alone (630 μg total protein; not plotted) with thehigh concentration of acceptor was 75 dpm [¹⁴C]GlcUA. The activity ofpmHS is greatly stimulated by exogenous acceptor.

FIG. 3 Gel Filtration Analysis of Radiolabeled Polymer Synthesized invitro. The crude membranes containing pmHS (0.7 mg total protein) wereincubated with UDP-[¹⁴C]GlcUA and UDP-[³H]GlcNAc (each 500 μM, 0.4 μCi)in a 200 μl reaction volume either in the presence (top panel) orabsence (bottom panel) of acceptor polymer (1 μg uronic acid). Aftervarious reaction times (denoted on curves: 20, 60, or 270 min), portionsof the samples (75%) were analyzed on the PolySep column (calibrationelution times in minutes: void volume, 9.8; 580 kDa dextran, 12.3; 145kDa dextran, 12.75, totally included volume, 16.7). The startingacceptor polymer eluted at 12.8 min. Large polymers composed of bothradiolabeled sugars (¹⁴C, C; ³H, H) in an equimolar ratio weresynthesized by pmHS.

FIGS. 4(A-D) graphically depicts the alignment of the pmHS (two clones:A2, B10) with PglA, KfiA, KfiC, and DcbF. pmHS is shown in variousforms: HSA1 and HSA2 are the two putative domains of pmHS; pORF=partialopen reading frame which was obtained before complete sequencedetermined; recon=reconstructed open reading frame with sequence frommultiple sources. FIG. 4A: KfiC, SEQ ID NO:29; HSA1, SEQ ID NO:30; KfiA,SEQ ID NO:7; HSA2, SEQ ID NO:31; Consensus, SEQ ID NO:32. FIG. 4B: pmHS,SEQ ID NO:2; pglA, SEQ ID NO:6; DcbF, SEQ ID NO:17; Consensus, SEQ IDNO:33. FIG. 4C: A2, SEQ ID NO:2; B10, SEQ ID NO:4; pglA, SEQ ID NO:6;DcbF, SEQ ID NO:17; Consensus, SEQ ID NO:34. FIG. 4D: pmHS, SEQ ID NO:2;pglA, SEQ ID NO:6; DcbF, SEQ ID NO:17; and Consensus, SEQ ID NO:35.

DETAILED DESCRIPTION OF THE INVENTION

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details of construction and the arrangement of the components setforth in the following description or illustrated in the drawings. Theinvention is capable of other embodiments or of being practiced orcarried out in various ways. Also, it is to be understood that thephraseology and terminology employed herein is for purpose ofdescription and should not be regarded as limiting.

As used herein, the term “nucleic acid segment” and “DNA segment” areused interchangeably and refer to a DNA molecule which has been isolatedfree of total genomic DNA of a particular species. Therefore, a“purified” DNA or nucleic acid segment as used herein, refers to a DNAsegment which contains a Heparin Synthase (“HS”) coding sequence yet isisolated away from, or purified free from, unrelated genomic DNA, forexample, total Pasteurella multocida or, for example, mammalian hostgenomic DNA. Included within the term “DNA segment”, are DNA segmentsand smaller fragments of such segments, and also recombinant vectors,including, for example, plasmids, cosmids, phage, viruses, and the like.

Similarly, a DNA segment comprising an isolated or purified pmHS(Pasteurella multocida Heparin Synthase) gene or a PglA gene refers to aDNA segment including HS coding sequences isolated substantially awayfrom other naturally occurring genes or protein encoding sequences. Inthis respect, the term “gene” is used for simplicity to refer to afunctional protein, polypeptide or peptide encoding unit. As will beunderstood by those in the art, this functional term includes genomicsequences, cDNA sequences or combinations thereof. “Isolatedsubstantially away from other coding sequences” means that the gene ofinterest, in this case pmHS or PglA, forms the significant part of thecoding region of the DNA segment, and that the DNA segment does notcontain large portions of naturally-occurring coding DNA, such as largechromosomal fragments or other functional genes or DNA coding regions.Of course, this refers to the DNA segment as originally isolated, anddoes not exclude genes or coding regions later added to, orintentionally left in the segment by the hand of man.

Due to certain advantages associated with the use of prokaryoticsources, one will likely realize the most advantages upon isolation ofthe HS genes from Pasteurella multocida. One such advantage is that,typically, eukaryotic enzymes may require significant post-translationalmodifications that can only be achieved in an eukaryotic host. This willtend to limit the applicability of any eukaryotic HS genes that areobtained. Additionally, such eukaryotic HS genes are dainty, fragile,and difficult, if not impossible, to transfer into prokaryotic hosts forlarge scale polymer production. Moreover, those of ordinary skill in theart will likely realize additional advantages in terms of time and easeof genetic manipulation where a prokaryotic enzyme gene is sought to beemployed. These additional advantages include (a) the ease of isolationof a prokaryotic gene because of the relatively small size of the genomeand, therefore, the reduced amount of screening of the correspondinggenomic library; and (b) the ease of manipulation because the overallsize of the coding region of a prokaryotic gene is significantly smallerdue to the absence of introns. Furthermore, if the product of the HSgenes (i.e., the enzyme) requires posttranslational modifications orcofactors, these would best be achieved in a similar prokaryoticcellular environment (host) from which the gene was derived.

Preferably, DNA sequences in accordance with the present invention willfurther include genetic control regions which allow the expression ofthe sequence in a selected recombinant host. Of course, the nature ofthe control region employed will generally vary depending on theparticular use (e.g., cloning host) envisioned.

In particular embodiments, the invention concerns isolated DNA segmentsand recombinant vectors incorporating DNA sequences which encode a HSgene such as pmHS or PglA. In the case of pmHS, the isolated DNAsegments and recombinant vectors incorporating DNA sequences whichinclude within their amino acid sequences an amino acid sequence inaccordance with SEQ ID NO:2 or SEQ ID NO:4; for PglA, an amino acidsequence in accordance with SEQ ID NO: 6. Moreover, in other particularembodiments, the invention concerns isolated DNA segments andrecombinant vectors incorporating DNA sequences which encode a gene thatincludes within its amino acid sequence the amino acid sequence of an HSgene or DNA, and in particular to a HS gene or cDNA, corresponding toPasteurella multocida Heparin Synthases—pmHS and PglA. For example,where the DNA segment or vector encodes a full length HS protein, or isintended for use in expressing the HS protein, preferred sequences arethose which are essentially as set forth in SEQ ID NO:2 or SEQ ID NO:4or SEQ ID NO:6. Additionally, sequences which have at least one or moreamino acid motifs (discussed in detail hereinafter) and encode afunctionally active heparosan/heparin synthase are contemplated for use.

The presently claimed and disclosed pmHS includes SEQ ID NOS:1 and 3(nucleotide sequence) and SEQ ID NOS:2 and 4 (amino acid sequences) thathave been assigned GenBank Accession Nos. AAL84702 and AAL84705,respectively. The presently claimed and disclosed PglA includes SEQ IDNO:5 (nucleotide sequence) and SEQ ID NO:6 (amino acid sequence) thathave been assigned GenBank Accession No. NP_(—)245351. Amino acid motifsfor enzymatically active heparosan/heparin synthases are disclosed indetail hereinafter.

Nucleic acid segments having heparin synthase activity may be isolatedby the methods described herein. The term “a sequence essentially as setforth in SEQ ID NO:2 or 4 or 6” means that the sequence substantiallycorresponds to a portion of SEQ ID NO:2 or 4 or 6 and has relatively fewamino acids which are not identical to, or a biologically functionalequivalent of, the amino acids of SEQ ID NO:2 or 4 or 6. The term“biologically functional equivalent” is well understood in the art andis further defined in detail herein, as a gene having a sequenceessentially as set forth in SEQ ID NO:2 or 4 or 6, and that isassociated with the ability of prokaryotes to produce heparin/heparosanor a “heparin like” polymer or a heparin synthase polypeptide. Forexample, PglA is approximately 70% identical to pmHS and PglA is shown,hereinafter, to be an enzymatically active heparin/heparosan synthase.

One of ordinary skill in the art would appreciate that a nucleic acidsegment encoding enzymatically active heparin synthase may containconserved or semi-conserved substitutions to the sequences set forth inSEQ ID NOS: 1, 2, 3, 4, 5 or 6 and yet still be within the scope of theinvention.

In particular, the art is replete with examples of practitioner'sability to make structural changes to a nucleic acid segment (i.e.encoding conserved or semi-conserved amino acid substitutions) and stillpreserve its enzymatic or functional activity. See for example: (1)Risler et al. “Amino Acid Substitutions in Structurally RelatedProteins. A Pattern Recognition Approach.” J. Mol. Biol. 204:1019-1029(1988) [“ . . . according to the observed exchangeability of amino acidside chains, only four groups could be delineated; (i) Ile and Val; (ii)Leu and Met, (iii) Lys, Arg, and Gln, and (iv) Tyr and Phe.”]; (2)Niefind et al. “Amino Acid Similarity Coefficients for Protein Modelingand Sequence Alignment Derived from Main-Chain Folding Anoles.” J. Mol.Biol. 219:481-497 (1991) [similarity parameters allow amino acidsubstitutions to be designed]; and (3) Overington et al.“Environment-Specific Amino Acid Substitution Tables: Tertiary Templatesand Prediction of Protein Folds,” Protein Science 1:216-226 (1992)[“Analysis of the pattern of observed substitutions as a function oflocal environment shows that there are distinct patterns . . . ”Compatible changes can be made]. Each of these articles, to the extentthat they provide additional details to one of ordinary skill in the artin the methods of making such conserved or semi-conserved amino acidsubstitutions, are hereby expressly incorporated herein in theirentirety as though set forth herein.

These references and countless others available to one of ordinary skillin the art, indicate that given a nucleic acid sequence, one of ordinaryskill in the art could make substitutions and changes to the nucleicacid sequence without changing its functionality. Also, a substitutednucleic acid segment may be highly identical and retain its enzymaticactivity with regard to its unadulterated parent, and yet still fail tohybridize thereto (i.e. spHAS and seHAS, 70% identical yet do nothybridize under standard hybridization conditions as definedhereinafter). Therefore, the ability of two sequences to hybridize toone another can be a starting point for comparison but should not be theonly ending point—rather, one of ordinary skill in the art must look tothe conserved and semi-conserved amino acid stretches between thesequences between the sequences and also must assess functionality.Thus, given that two sequences may have conserved and/or semi-conservedamino acid stretches, functionality must be assessed.

One of ordinary skill in the art would also appreciate thatsubstitutions can be made to the pmHS nucleic acid segment listed in SEQID NO: 1 or 3 that do not affect the amino acid sequences they encode orresult in conservative or semi-conservative substitutions in the aminoacid sequences they encode without deviating outside the scope andclaims of the present invention. Standardized and accepted functionallyequivalent amino acid substitutions are presented in Table I.

TABLE I Conservative and Semi- Amino Acid Group ConservativeSubstitutions NonPolar R Groups Alanine, Valine, Leucine, Isoleucine,Proline, Methionine, Phenylalanine, Tryptophan Polar, but uncharged, RGroups Serine, Threonine, Cysteine, Asparagine, Glutamine NegativelyCharged R Groups Aspartic Acid, Glutamic Acid Positively Charged RGroups Lysine, Arginine, Histidine

A particular example would be SEQ ID NO NOS: 2 and 4, both of whichencode a functionally active HS and yet have a single substitution atposition 455 (Threonine for Isoleucine), and yet both enzymes are stillcapable of producing heparosan. Such a conservative or semi-conservativescheme is even more evident when comparing pmHS with PglA—they are only˜70% identical and yet still both produce functionally active HSenzymes.

Another preferred embodiment of the present invention is a purifiednucleic acid segment that encodes a protein in accordance with SEQ IDNO:2 or 4 or 6 further defined as a recombinant vector. As used herein,the term “recombinant vector” refers to a vector that has been modifiedto contain a nucleic acid segment that encodes a HS protein, or fragmentthereof. The recombinant vector may be further defined as an expressionvector comprising a promoter operatively linked to said HS encodingnucleic acid segment.

A further preferred embodiment of the present invention is a host cell,made recombinant with a recombinant vector comprising a HS gene. Thepreferred recombinant host cell may be a prokaryotic cell. In anotherembodiment, the recombinant host cell is an eukaryotic cell. As usedherein, the term “engineered” or “recombinant” cell is intended to referto a cell into which a recombinant gene, such as a gene encoding HS, hasbeen introduced. Therefore, engineered cells are distinguishable fromnaturally occurring cells which do not contain a recombinantlyintroduced gene. Engineered cells are thus cells having a gene or genesintroduced through the hand of man. Recombinantly introduced genes willeither be in the form of a cDNA gene, one or more copies of a genomicgene, or will include genes positioned adjacent to a promoter notnaturally associated with the particular introduced gene.

Where one desires to use a host other than Pasteurella, as may be usedto produce recombinant heparin/heparosan synthase, it may beadvantageous to employ a prokaryotic system such as E. coli, B.subtilis, Lactococcus sp., (see, for example, U.S. patent applicationSer. No. 09/469,200, which discloses the production of HA through theintroduction of a HAS gene into Bacillus host—the contents of which areexpressly incorporated herein in their entirety), or even eukaryoticsystems such as yeast or Chinese hamster ovary, African green monkeykidney cells, VERO cells, or the like. Of course, where this isundertaken it will generally be desirable to bring the heparin/heparosansynthase gene under the control of sequences which are functional in theselected alternative host. The appropriate DNA control sequences, aswell as their construction and use, are generally well known in the artas discussed in more detail hereinbelow.

In preferred embodiments, the heparin/heparosan synthase-encoding DNAsegments further include DNA sequences, known in the art functionally asorigins of replication or “replicons”, which allow replication ofcontiguous sequences by the particular host. Such origins allow thepreparation of extrachromosomally localized and replicating chimericsegments or plasmids, to which HS DNA sequences are ligated. In morepreferred instances, the employed origin is one capable of replicationin bacterial hosts suitable for biotechnology applications. However, formore versatility of cloned DNA segments, it may be desirable toalternatively or even additionally employ origins recognized by otherhost systems whose use is contemplated (such as in a shuttle vector).

The isolation and use of other replication origins such as the SV40,polyoma or bovine papilloma virus origins, which may be employed forcloning or expression in a number of higher organisms, are well known tothose of ordinary skill in the art. In certain embodiments, theinvention may thus be defined in terms of a recombinant transformationvector which includes the HS coding gene sequence together with anappropriate replication origin and under the control of selected controlregions.

Thus, it will be appreciated by those of ordinary skill in the art thatother means may be used to obtain the HS gene or cDNA, in light of thepresent disclosure. For example, polymerase chain reaction or RT-PCRproduced DNA fragments may be obtained which contain full complements ofgenes or cDNAs from a number of sources, including other strains ofPasteurella or from eukaryotic sources, such as cDNA libraries.Virtually any molecular cloning approach may be employed for thegeneration of DNA fragments in accordance with the present invention.Thus, the only limitation generally on the particular method employedfor DNA isolation is that the isolated nucleic acids should encode abiologically functional equivalent HS, and in a more preferredembodiment, the isolated nucleic acids should encode an amino acidsequence that contains at least one of the HS amino acid motifsdescribed in detail hereinafter.

Once the DNA has been isolated it is ligated together with a selectedvector. Virtually any cloning vector can be employed to realizeadvantages in accordance with the invention. Typical useful vectorsinclude plasmids and phages for use in prokaryotic organisms and evenviral vectors for use in eukaryotic organisms. Examples includepKK223-3, pSA3, recombinant lambda, SV40, polyoma, adenovirus, bovinepapilloma virus and retroviruses. However, it is believed thatparticular advantages will ultimately be realized where vectors capableof replication in both Lactococcus or Bacillus strains and E. coli or P.multocida are employed.

Vectors such as these, exemplified by the pSA3 vector of Dao andFerretti or the pAT19 vector of Trieu-Cuot, et al., allow one to performclonal colony selection in an easily manipulated host such as E. coil,followed by subsequent transfer back into a food grade Lactococcus orBacillus strain for production of heparin/heparosan. These are benignand well studied organisms used in the production of certain foods andbiotechnology products—otherwise known in the art as GRAS (GenerallyRegarded As Safe). GRAS organisms are advantageous in that one canaugment the Lactococcus or Bacillus strain's ability to synthesizeheparin/heparosan through gene dosaging (i.e., providing extra copies ofthe heparosan synthase gene by amplification) and/or the inclusion ofadditional genes to increase the availability of the heparin/heparosanprecursors UDP-GlcUA and UDP-GlcNAc and/or the inclusion of genes thatinclude enzymes that wilt make modifications (such as sulfation andepimerization) to the heparosan polymer in order to convert it toheparin. Sugar precursors are made by the enzymes with UDP-glucosedehydrogenase and UDP-N-acetylglucosamine pyrophosphorylase activity,respectively. The inherent ability of a bacterium to synthesizeheparin/heparosan is also augmented through the formation of extracopies, or amplification, of the plasmid that carries theheparin/heparosan synthase gene. This amplification can account for upto a 10-fold increase in plasmid copy number and, therefore, the HS genecopy number.

Another procedure that would further augment HS gene copy number is theinsertion of multiple copies of the gene into the plasmid. Anothertechnique would include integrating the HS gene into chromosomal DNA.This extra amplification would be especially feasible, since the HS genesize is small. In some scenarios, the chromosomal DNA-ligated vector isemployed to transfect the host that is selected for clonal screeningpurposes such as E. coli or Bacillus, through the use of a vector thatis capable of expressing the inserted DNA in the chosen host. In certaininstances, especially to confer stability, genes such as the HS gene,may be integrated into the chromosome in various positions in anoperative fashion. Unlike plasmids, integrated genes do not needselection pressure for maintenance of the recombinant gene.

Where an eukaryotic source such as dermal or synovial fibroblasts orrooster comb cells is employed, one will desire to proceed initially bypreparing a cDNA library. This is carried out first by isolation of mRNAfrom the above cells, followed by preparation of double stranded cDNAand ligation of the cDNA with the selected vector. Numerouspossibilities are available and known in the art for the preparation ofthe double stranded cDNA, and all such techniques are believed to beapplicable. A preferred technique involves reverse transcriptionutilizing an enzyme having reverse transcriptase activity. Once apopulation of double stranded cDNAs is obtained, a cDNA library isprepared in the selected host by accepted techniques, such as byligation into the appropriate vector and amplification in theappropriate host. Due to the high number of clones that are obtained,and the relative ease of screening large numbers of clones by thetechniques set forth herein, one may desire to employ phage expressionvectors, such as λgt11, λgt12, λGem11, and/or λZAP for the cloning andexpression screening of cDNA clones.

In certain other embodiments, the invention concerns isolated DNAsegments and recombinant vectors that include within their sequence anucleic acid sequence essentially as set forth in SEQ ID NO:1 or 3 or 5.The term “essentially as set forth in SEQ ID NO:1 or 3 or 5” is used inthe same sense as described above with respect to the amino acidsequences and means that the nucleic acid sequence substantiallycorresponds to a portion of SEQ ID NO:1 or 3 or 5, and has relativelyfew codons that are not identical, or functionally equivalent, to thecodons of SEQ ID NO:1 or 3 or 5 and encodes a enzymatically active HS.The term “functionally equivalent codon” is used herein to refer tocodons that encode the same amino acid, such as the six codons forarginine or serine, and also refers to codons that encode biologicallyequivalent amino acids. “Biologically Equivalent Amino Acids” of Table Irefers to residues that have similar chemical or physical propertiesthat may be easily interchanged for one another.

It will also be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids or 5′ or 3′ nucleic acid sequences, and yet still beessentially as set forth in one of the sequences disclosed herein, solong as the sequence meets the criteria set forth above, including themaintenance of biological protein activity where protein expression andenzymatic activity is concerned. The addition of terminal sequencesparticularly applies to nucleic acid sequences which may, for example,include various non-coding sequences flanking either of the 5′ or 3′portions of the coding region or may include various internal sequences,which are known to occur within genes.

Likewise, deletion of certain portions of the polypeptide can bedesirable. For example, functional truncated versions of pmHAS, thePasteurella hyaluronan synthase, missing the carboxyl terminus enhancesthe utility for in vitro use. The truncated pmHAS enzyme is a solubleprotein that is easy to purify in contrast to the full-length protein(972 residues). Also, expression level of the enzyme increases greatlyas the membrane is not overloaded. It is also contemplated that atruncated version of pmHS would also be useful and is contemplated asfalling within the scope of the presently claimed and disclosedinvention. Such a truncated version would also be highly soluble andincrease expression of the enzyme; the native membrane proteins arefound in low levels and are not soluble without special treatment withdetergents.

Allowing for the degeneracy of the genetic code as well as conserved andsemi-conserved substitutions, sequences which have between about 40% andabout 80%; or more preferably, between about 80% and about 90%; or evenmore preferably, between about 90% and about 99% of nucleotides whichare identical to the nucleotides of SEQ ID NO:1 or 3 or 5 will besequences which are “essentially as set forth in SEQ ID NO:1 or 3 or 5”.In a preferred embodiment, the sequences would be 70% identical.Sequences which are essentially the same as those set forth in SEQ IDNO:1 or 3 or 5 may also be functionally defined as sequences which arecapable of hybridizing to a nucleic acid segment containing thecomplement of SEQ ID NO:1 or 3 or 5 under standard or less stringenthybridizing conditions. Suitable standard hybridization conditions willbe well known to those of skill in the art and are clearly set forthhereinbelow. As certain domains and active sites are formed from arelatively small portion of the total polypeptide, these regions ofsequence identity or similarity may be present only in portions of thegene. Additionally, sequences which are “essentially as set forth in SEQID NO:1 or 3 or 5” will include those amino acid sequences that have atleast one of the amino acid motifs (described hereinafter in detail) andthat also retain the functionality of an enzymatically active HS.

As is well known to those of ordinary skill in the art, most of theamino acids in a protein are present to form the “scaffolding” orgeneral environment of the protein. The actual working parts responsiblefor the specific desired catalysis are usually a series of small domainsor motifs. Thus, a pair of enzymes that possess the same or similarmotifs would be expected to possess the same or similar catalyticactivity, thus they are functionally equivalent. Utility for thishypothetical pair of enzymes may be considered interchangeable unlessone member of the pair has a subset of distinct, useful properties. In asimilar vein, certain non-critical motifs or domains may be dissectedfrom the original, naturally occurring protein and function will not beaffected; removal of non-critical residues does not perturb theimportant action of the remaining critical motifs or domains. Byanalogy, with sufficient planning and knowledge, it is possible totranslocate motifs or domains from one enzyme to another polypeptide toconfer the new enzyme with desirable characteristics intrinsic to thedomain or motif. Such motifs for HS are disclosed in particularlyhereinafter.

The term “standard hybridization conditions” as used herein, is used todescribe those conditions under which substantially complementarynucleic acid segments will form standard Watson-Crick base-pairing. Anumber of factors are known that determine the specificity of binding orhybridization, such as pH, temperature, salt concentration, the presenceof agents such as formamide and dimethyl sulfoxide, the length of thesegments that are hybridizing, and the like. When it is contemplatedthat shorter nucleic acid segments will be used for hybridization, forexample fragments between about 14 and about 100 nucleotides, salt andtemperature preferred conditions for overnight standard hybridizationwill include 1.2-1.8×HPB (High Phosphate Buffer) at 40-50° C. or 5×SSC(Standard Saline Citrate) at 50° C. Washes in low salt (10 mM salt or0.1×SSC) are used for stringent hybridizations with room temperatureincubations of 10-60 minutes. Washes with 0.5× to 1×SSC, 1% Sodiumdodecyl sulfate at room temperature are used in lower stringency washesfor 15-30 minutes. For all hybridizations: (where 1×HPB=0.5 m NaCl, 0.1m Na₂HPO₄, 5 mM EDTA, pH 7.0) and (where 20×SSC=3 m NaCl, 0.3 m SodiumCitrate with pH 7.0).

Naturally, the present invention also encompasses DNA segments which arecomplementary, or essentially complementary, to the sequence set forthin SEQ ID NOS:1, 2, 3, 4, 5, or 6. Nucleic acid sequences which are“complementary” are those which are capable of base-pairing according tothe standard Watson-Crick complementarity rules. As used herein, theterm “complementary sequences” means nucleic acid sequences which aresubstantially complementary, as may be assessed by the same nucleotidecomparison set forth above, or as defined as being capable ofhybridizing to the nucleic acid segment of SEQ ID NO:1, 2, 3, 4, 5, or 6under the above-defined standard hybridization conditions.

The nucleic acid segments-of the present invention, regardless of thelength of the coding sequence itself, may be combined with other DNAsequences, such as promoters, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, epitope tags, polyhistidine regions, other coding segments, and the like, such that theiroverall length may vary considerably. For example, functionalspHAS-(Histidine)₆ and x1HAS1-(Green Fluorescent Protein) fusionproteins have been reported. It is therefore contemplated that a nucleicacid fragment of almost any length may be employed, with the totallength preferably being limited by the ease of preparation and use inthe intended recombinant DNA protocol.

Naturally, it will also be understood that this invention is not limitedto the particular nucleic acid and amino acid sequences of SEQ ID NOS:1,2, 3, 4, 5, or 6. Recombinant vectors and isolated DNA segments maytherefore variously include the HS coding regions themselves, codingregions bearing selected alterations or modifications in the basiccoding region, or they may encode larger polypeptides which neverthelessinclude HS coding regions or may encode biologically functionalequivalent proteins or peptides which have variant amino acid sequences.

The DNA segments of the present invention encompass biologicallyfunctional equivalent HS proteins and peptides. Such sequences may ariseas a consequence of codon redundancy and functional equivalency whichare known to occur naturally within nucleic acid sequences and theproteins thus encoded. Alternatively, functionally equivalent proteinsor peptides may be created via the application of recombinant DNAtechnology, in which changes in the protein structure may be engineered,based on considerations of the properties of the amino acids beingexchanged. Changes designed by man may be introduced through theapplication of site-directed mutagenesis techniques, e.g., to introduceimprovements to the enzyme activity or to antigenicity of the HS proteinor to test HS mutants in order to examine HS activity at the molecularlevel.

Also, specific changes to the HS coding sequence will result in theproduction of heparin/heparosan having a modified size distribution orstructural configuration. One of ordinary skill in the art wouldappreciate that the HS coding sequence can be manipulated in a manner toproduce an altered HS which in turn is capable of producingheparin/heparosan having differing polymer sizes and/or functionalcapabilities. The utility of such a modified polymer is easilyappreciated from the above “Background of the Invention.” For example,the HS coding sequence may be altered in such a manner that the HS hasan altered sugar substrate specificity so that the HS creates a newheparin/heparosan-like chimeric polymer incorporating a differentstructure via the inclusion of a previously unincorporated sugar orsugar derivative. This newly incorporated sugar results in a modifiedheparin/heparosan having different and unique functional properties. Aswill be appreciated by one of ordinary skill in the art given the HScoding sequences, changes and/or substitutions can be made to the HScoding sequence such that these desired properties and/or sizemodifications can be accomplished.

Basic knowledge on the substrate binding sites (e.g. the UDP-GlcUA siteor UDP-GlcNAc site or oligosaccharide acceptor site) of pmHS or pglAallows the targeting of residues for mutation to change the catalyticproperties of the site. The identity of important catalytic residues ofpmHAS, another GAG synthase, have recently been elucidated (Jing &DeAngelis, 2000, Glycobiology vol 10; pp. 883-889 the contents of whichare expressly incorporated herein in their entirety). Appropriatechanges at or near these residues alters UDP-sugar binding. Changes ofresidues in close proximity should allow other precursors to bindinstead of the authentic heparin/heparosan sugar precursors; thus a new,modified polymer is synthesized. Polymer size changes are caused bydifferences in the synthase's catalytic efficiency or changes in theacceptor site affinity. Polymer size changes have been made in seHAS andspHAS (U.S. patent application Ser. Nos. 09/559,793 and 09/469,200, thecontents of which are expressly incorporated herein by reference) aswell as the vertebrate HAS, xlHAS1 (DG42) (Pummill & DeAngelis, in pressand which is also incorporated herein in its entirety) by mutatingvarious residues. As pmHS is a more malleable, robust enzyme than theseother enzymes, similar or superior versions of mutant pmHS or pglA whichsynthesize modified polymers are easily produced.

The term “modified structure” as used herein denotes a heparin/heparosanpolymer containing a sugar or derivative not normally found in thenaturally occurring heparin/heparosan polypeptide. The term “modifiedsize distribution” refers to the synthesis of heparin/heparosanmolecules of a size distribution not normally found with the nativeenzyme; the engineered size could be much smaller or larger than normal.

One of ordinary skill in the art given this disclosure would appreciatethat there are several ways in which the size distribution of theheparin/heparosan polymer made by the HS could be regulated to givedifferent sizes. First, the kinetic control of product size can bealtered by environmental factors such as decreasing temperature,decreasing time of enzyme action and/or by decreasing the concentrationof one or both sugar nucleotide substrates. Decreasing any or all ofthese variables will give lower amounts and smaller sizes ofheparin/heparosan product. The disadvantages of these extrinsicapproaches are that the yield of product is also decreased and it isdifficult to achieve reproducibility from day to day or batch to batch.Secondly, the alteration of the intrinsic ability of the enzyme tosynthesize a large or small heparin/heparosan product. Changes to theprotein are engineered by recombinant DNA technology, includingsubstitution, deletion and addition of specific amino acids (or even theintroduction of prosthetic groups through metabolic processing). Suchchanges that result in an intrinsically slower enzyme then allow formore reproducible control of heparin/heparosan size by kinetic means.The final heparin/heparosan size distribution is determined by certaincharacteristics of the enzyme that rely on particular amino acids in thesequence. Among the residues absolutely conserved between the now knownHS enzymes, there is a set of amino acids at unique positions thatcontrol or greatly influence the size of the polymer that the enzyme canmake.

Finally, using post-synthesis processing larger molecular weight heparincan be degraded with specific glycasidases or ultrasonication, acids ora combination thereof to make lower molecular weight heparin/heparosan.This practice, however, is very difficult to achieve reproducibility andone must meticulously repurify the heparin/heparosan to remove thecleavage reagent and unwanted digestion products.

Structurally modified heparin/heparosan is no different conceptuallythan altering the size distribution of the heparin/heparosan product bychanging particular amino acids in the desired HS and/or moreparticularly, but not limiting thereto, pmHS or PglA. Derivatives ofUDP-GlcNAc, in which the acetyl group is missing from the amine(UDP-GlcN) or replaced with another chemically useful group (forexample, phenyl to produce UDP-GlcNPhe), are expected to be particularlyuseful. The free amino group would be available for chemical reactionsto derivatize heparin/heparosan in the former case with GlcNincorporation. In the latter case, GlcNPhe, would make the polymer morehydrophobic or prone to making emulsions. The strong substratespecificity may rely on a particular subset of amino acids among theresidues that are conserved. Specific changes to one or more of theseresidues creates a functional HS that interacts less specifically withone or more of the substrates than the native enzyme. This alteredenzyme then utilizes alternate natural or special sugar nucleotides toincorporate sugar derivatives designed to allow different chemistries tobe employed for the following purposes: (i) covalently coupling specificdrugs, proteins, or toxins to the structurally modifiedheparin/heparosan for general or targeted drug delivery, radiologicalprocedures, etc. (ii) covalently cross linking the heparin/heparosanitself or to other supports to achieve a gel, or other three dimensionalbiomaterial with stronger physical properties, and (iii) covalentlylinking heparin/heparosan to a surface to create a biocompatible film ormonolayer.

Experimental

As stated hereinabove, Pasteurella multocida Type D, a causative agentof atrophic rhinitis in swine and pasteurellosis in other domesticanimals, produces an extracellular polysaccharide capsule that is aputative virulence factor. It has been reported that the capsule of TypeD was removed by treating microbes with heparin lyase III. A 617-residueenzyme, pmHS, and a 651-residue enzyme, PglA, which are both authenticheparosan (unsulfated, unepimerized heparin) synthase enzymes have beenmolecularely cloned and are presently claimed and disclosed herein.Recombinant Escherichia coli-derived pmHS or PglA catalyzes thepolymerization of the monosaccharides from UDP-GlcNAc and UDP-GlcUA.Other structurally related sugar nucleotides do not substitute. Synthaseactivity was stimulated about 7- to 25-fold by the addition of anexogenous polymer acceptor. Molecules composed of ˜500 to 3,000 sugarresidues were produced in vitro. The polysaccharide was sensitive to theaction of heparin lyase III but resistant to hyaluronan lyase. Thesequence of pmHS enzyme is not very similar to the vertebrateheparin/heparan sulfate glycosyltransferases, EXT1/2, or to otherPasteurella glycosaminoglycan synthases that produce hyaluronan orchondroitin. Certain motifs do exist however, between the pmHS, pglA,and KfiA and KfiC thereby leading to deduced amino acid motifs that areconserved throughout this class of GAG synthases for the production ofheparin/heparosan The pmHS enzyme is the first microbial dual-actionglycosyltransferase to be described that forms a polysaccharide composedof β4GlcUA-α4GlcNAc disaccharide repeats. In contrast, heparosanbiosynthesis in E. coli K5 requires at least two separate polypeptides,KfiA and KfiC, to catalyze the same polymerization reaction.

Glycosaminoglycans [GAGs] are long linear polysaccharides consisting ofdisaccharide repeats that contain an amino sugar ⁽¹⁻²⁾. Heparin/heparan[β4GlcUA-α4GlcNAc]_(n), chondroitin [β4GlcUA-β3GalNAc]_(n), andhyaluronan [β4GlcUA-β3GlcNAc]_(n) are three prevalent GAGs and the onlyknown acidic GAGs. In the former two polymers, usually n=20 to 100 whilein the case of HA, typically n=103-4. In vertebrates, one or moremodifications including O-sulfation of certain hydroxyls, deacetylationand subsequent N-sulfation, or epimerization of glucuronic acid toiduronic acid are found in most GAGs except HA⁽³⁾. A few clever microbesalso produce GAG chains, however, sulfation or epimerization have notbeen described. The GAGs in pathogenic bacteria are found asextracellular polysaccharide coatings, called capsules, which serve asvirulence factors⁽⁴⁾. The capsule is thought to assist in the evasion ofhost defenses such as phagocytosis and complement. As the microbialpolysaccharide is identical or very similar to the host GAG, theantibody response is either very limited or non-existent.

The invasiveness and pathogenicity of certain Escherichia coli strainshas been attributed to their polysaccharide capsule⁽⁴⁾. Two E. colicapsular types, K5 and K4, make polymers composed of GAG-like polymers.The K5 capsular material is a polysaccharide called heparosan,N-acetylheparosan, or desulfoheparin, which are identical to mammalianheparin/heparin sulfate except that the bacterial polymer is unsulfatedand there is no epimerization of GlcUA to iduronic acid ⁽⁵⁾. The E. coliK4 polymer is an unsulfated chondroitin backbone decorated with fructoseside-branches on the C3 position of the GlcUA residues⁽⁶⁾.

The E. coli K5 capsule biosynthesis locus contains the open readingframes KfiA-D (also called Kfa in some reports; GenBank Accession NumberX77617). At first, KfiC was stated to be a dual-actionglycosyltransferase responsible for the alternating addition of bothGlcUA and GlcNAc to the heparosan chain⁽⁷⁾. However, a later report bythe same group reported that another protein, KfiA, was actually theαGlcNAc-transferase required for Heparosan polymerization ⁽⁸⁾.Therefore, at least these two enzymes, KfiA and KfiC, theβGlcUA-transferase, work in concert to form the disaccharide repeat andthe first report, that KfiC was a dual-action enzyme, was in error.Another deduced protein in the operon, KfiB, was suggested to stabilizethe enzymatic complex during elongation in vivo, but perhaps notparticipate directly in catalysis ⁽⁸⁾. The identity and the sequence ofthe E. coli K4 capsular glycosyltransferase(s) has recently beenreported. This enzyme, KfoC, is approximately 60% identical to thePasteurella chondroitin synthase (pmCS) and is also a dual-actionenzyme.

Many P. multocida isolates produce GAG or GAG-like molecules as assessedby enzymatic degradation and removal of the capsule of living bacterialcells^((9,10)). Carter Type A P. multocida, the major causative agent offowl cholera and pasteurellosis, makes an HA capsule⁽¹¹⁾. A singlepolypeptide, the HA synthase, pmHAS, polymerizes the HA chain bytransferring both GlcUA and GlcNAc ⁽¹²⁾. Type F P. multocida, the minorfowl cholera pathogen, produces a capsule composed of an unsulfatedchondroitin sensitive to Flavobacterium chondroitin AClyase^((9,13,14)). Again, a dual-action chondroitin synthase, pmCS,polymerizes the chondroitin chain⁽¹⁴⁾. The capsule of another distinctP. multocida, Type D, was reported to be sensitive to heparin lyase III⁽⁹⁾ which thereby led to the presently claimed and disclosedinvention—the identification and characterization of pmHS (P. multocidaheparin/heparosan synthase) and PglA, the first and only known bacterialdual-action heparosan synthases.

Prior to recombinantly obtaining the pmHS gene and heterologouslyexpressing it in a recombinant system, activity assays of P. multocidaType D enzymes were completed. Native membranes were prepared from awild-type encapsulated Type D strain (P-3881; DeAngelis et al., 1996,the entirety of which is expressly incorporated herein in its entirety).The membranes were tested for in vitro sugar incorporation monitored bypaper chromatography analysis. Characterization of the ability toco-polymerize the two sugars and utilize metal ions was performed.First, detection of co-polymerization activity of the Type D P.multocida strain was determined in vitro. The membranes+UDP-[¹⁴C]GlcUA(300 μM; 1.5×10⁵ dpm)+various combinations of the 2^(nd) sugar(UDP-GlcNAc, 900 μM) and/or EDTA chelator (45 mM) were mixed in 50 mMTris, pH 7.2 with 20 mM MnCl₂ and 20 mM MgCl₂ reaction buffer. Allreactions were performed at 30 degrees Celsius for 2.5 hours. Theincorporation was measured by paper chromatography as disclosed inDeAngelis et al., 1996. The results of this co-polymerization activityare summarized in Table II.

TABLE II UDP-GlcNAc Added? EDTA Added? Incorporation (dpm) No No 520 YesNo 9150 No Yes 35 Yes Yes 160

Thus, it is apparent that the Type D P. multocida strain P-3881 has ametal-dependent enzyme that copolymerized both heparin precursors into apolymer.

Second, the metal requirement of the Type D P. multocida HS activity wastested in vitro. Membranes+UDP-[¹⁴C]GlcUA+UDP-GlcNAc and buffer withoutthe metals were mixed in a similar fashion as the preceding experimentexcept that various metals or EDTA (20 mM) were added as noted in TableIII. The results of this metal specificity are summarized in Table III.

TABLE III Metal dpm None 13 Mg 2960 Mn 3070 Mn + Mg 3000 Co 120

Thus, it is apparent that the Type D P. multocida HS requires eithermanganese or magnesium ion for enzymatic activity.

Further, the sugar specificity of the Type D P. multocida strain wasdetermined in vitro in similar experiments. The ability to co-polymerizethe sugars that compose the authentic backbone was tested by performingtwo parallel reactions:

-   A. UDP-[¹⁴C]GlcUA+various combinations of 2^(nd) UDP-sugars.-   B. UDP-[³H]GlcNAc+various combinations of 2^(nd) UDP-sugars    The results of these experiments are summarized in Table IV.    Significant ¹⁴C-GlcUA incorporation required UDP-GlcNAc and,    conversely, significant ³H-GlcNAc incorporation required UDP-GlcUA;    the enzyme copolymerizes the polysaccharide chain with both    authentic heparin UDP-sugar precursors.

TABLE IV A. Hexosamine-transfer 2^(nd) Sugar Added ¹⁴C dpm incorporationNone 330 UDP-GlcNAc 2290 UDP-GalNAc 2790 UDP-Glc 450 B. Uronic AcidTransfer 2^(nd) Sugar Added ³H dpm incorporation None 170 UDP-GlcUA 1000UDP-GalUA 290 UDP-Glc 185

It should be added that the above-described results show that the nativeType D P. multocida membrane enzymes have relaxed hexosamine transferspecificity in vitro. Such relaxed hexosamine transfer specificity is anadvantage for syntheses where the UDP-sugar supplied can be manipulated.In such a manner, novel and non-naturally occurring polymers can becreated. These novel, non-naturally occurring polymers have significantutility and novel biological properties.

Experimental Procedures for Isolating HS Genes and Testing Function

Materials and Pasteurella Strains—Unless otherwise noted, all chemicalswere from Sigma or Fisher, and all molecular biology reagents were fromPromega. The wild-type encapsulated Type D P. multocida isolates, P-934(swine), P-3881 (bovine), P-4058 (rabbit), and P-5695 (swine), wereobtained from the USDA collection (Ames, Iowa). The strains were grownin brain heart infusion (Difco) at 37° C.

Analysis of Genomic DNA and Isolation of Capsule Biosynthesis LocusDNA—Preliminary data from Southern blot analysis using pmHAS-basedhybridization probes (12) suggested that the Type A synthase and theputative Type D synthase were not very similar at the DNA level.However, PCR suggested that the UDP-glucose dehydrogenase genes, whichencode an enzyme that produces the UDP-GlcUA precursor required for bothHA and heparin biosynthesis, were very homologous. In most encapsulatedbacteria, the precursor-forming enzymes and the transferases are locatedin the same operon. To make a hybridization probe predicted to detectthe capsule locus, Type D chromosomal DNA served as a template in PCRreactions utilizing degenerate oligonucleotide primers (sense:GARTTYBTIMRIGARGGIAARGCIYTITAYGAY (SEQ ID NO:12); antisense:RCARTAICCICCRTAICCRAAISWXGGRTTRTTRTARTG (SEQ ID NO:13), where I=inosine;R=A or G; S=C or G; W=A or T; Y=C or T) corresponding to a conservedcentral region in many known UDP-glucose dehydrogenase genes. The˜0.3-kb amplicon was generated using Taq DNA polymerase (Fisher),gel-purified, and labeled with digoxigenin (High Prime system,Boehringer Mannheim).

A lambda library of Sau3A partially digested P-3881 DNA (˜4-9 kb averagelength insert) was made using the BamHI-cleaved λZap Express™ vectorsystem (Stratagene). The plaque lifts were screened by hybridization(5×SSC, 50° C.; 16 hrs) with the digoxigenin-labeled probe using themanufacturer guidelines for calorimetric development. E. coli XLI-BlueMRF′ was co-infected with the purified, individual positive lambdaclones and ExAssist helper phage to yield phagemids. The resultingphagemids were transfected into E. coli XLOLR cells to recover theplasmids. Sequence analysis of the plasmids using a variety of customprimers as well as the GPS-1 Genome Priming System (New England Biolabs)revealed a novel open reading frame, which we called pmHS (DNA sequencefacilities at Oklahoma State University and University of Oklahoma HSC).We amplified and sequenced the ORF from several highly encapsulatedisolates (see hereinbelow); very similar sequences were obtained.

Expression of Recombinant P. multocida Heparosan Synthase—The pmHS ORF(617 amino acids) was amplified from the various Type D genomic DNAtemplate by 18 cycles of PCR (16) with Taq polymerase. For constructingthe full-length enzyme, the sense primer (ATGAGCTTATTTAAACGTGCTACTGAGC(SEQ ID NO:14)) corresponded to the sequence at the deduced aminoterminus of the ORF and the antisense primer(TTTACTCGTTATAAAAAGATAAACACGGAATAAG (SEQ ID NO:15)) encoded the carboxylterminus including the stop codon. In addition, a truncated version ofpmHS was produced by PCR with the same sense primer but a differentantisense primer (TATATTTACAGCAGTATCATTTTCTAAAGG (SEQ ID NO:16)) toyield a predicted 501-residue protein, DcbF (SEQ ID NO:17) (GenBankAccession Number AAK17905)¹⁵; this variant corresponds to residues 1-497of pmHS followed by the residues TFRK.

The amplicons were cloned using the pETBlue-1 Acceptor system (Novagen)according to the manufacturer's instructions. The Taq-generated single Aoverhang is used to facilitate the cloning of the open reading framedownstream of the T7 promoter and the ribosome binding site of thevector. The ligated products were transformed into E. coli NovaBlue andplated on LB carbenicillin (50 μg/ml) and tetracycline (13 μg/ml) underconditions for blue/white screening. White colonies were analyzed byPCR-based screening and by restriction digestion. Plasmids with thedesired ORF were transformed into E. coli Tuner, the T7 RNApolymerase-containing expression host, and maintained on LB media withcarbenicillin and chloramphenicol (34 μg/ml) at 30° C. Mid-log phasecultures were induced with β-isopropylthiogalactoside (0.2 mM final) for5 hrs. The cells were harvested by centrifugation, frozen, and membraneswere prepared according to a cold lysozyme/sonication method¹⁶ except0.1 mM mercaptoethanol was included during the sonication steps.Membrane pellets were suspended in 50 mM Tris, pH 7.2, 0.1 mM EDTA andprotease inhibitors.

Assays for Heparosan Synthase Activity—Incorporation of radiolabeledmonosaccharides from UDP-[¹⁴C]GlcUA and/or UDP-[³H]GlcNAc precursors(NEN) was used to monitor heparosan synthase activity. Samples wereassayed in a buffer containing 50 mM Tris, pH 7.2, 10 mM MgCl₂₁ 10 mMMnCl₂, 0-0.6 mM UDP-GlcUA, and 0-0.6 mM UDP-GlcNAc at 30° C. Dependingon the experiment, a Type D acceptor polymer processed by extendedultrasonication of a capsular polysaccharide preparation (isolated bycetylpyridinium chloride precipitation of the spent Type D culturemedia)¹⁴ was also added to the reaction mixture. The reaction productswere separated from substrates by descending paper (Whatman 3M)chromatography with ethanol/1 M ammonium acetate, pH 5.5, developmentsolvent (65:35). The origin of the paper strip was cut out, eluted withwater, and the incorporation of radioactive sugars into polymer wasdetected by liquid scintillation counting with BioSafe II cocktail(RPI).

The metal preference of pmHS was assessed by comparing the signal from a“no metal” control reaction (0.5 mM EDTA) to reactions containing 10 to20 mM manganese, magnesium, or cobalt chloride. To test the transferspecificity of pmHS, various UDP-sugars (UDP-GalNAc, UDPGalUA, orUDP-Glc) were substituted for the authentic heparosan precursors. Thedata from the recombinant construct containing pmHS gene from the P-4058strain is presented, but the results were similar to constructs derivedfrom the P-934 or P-5695 strains.

Size Analysis and Enzymatic Degradation of Labeled Polymers—Gelfiltration chromatography was used to analyze the size distribution ofthe labeled polymers. Separations were performed with a Polysep-GFC-P4000 column (300×7.8 mm; Phenomenex) eluted with 0.2 M sodium nitrate at0.6 ml/min. Radioactivity was monitored with an in-line Radioflow LB508detector (EG & G Berthold; 500 μl flow cell) using Unisafe I cocktail(1.8 ml/min; Zinsser). The column was standardized withfluorescein-labeled dextrans of various sizes. To further characterizethe radiolabeled polymers, depolymerization tests with specificglycosidases was performed. The high molecular weight product waspurified by paper chromatography. The origin of the strips was washedwith 80% ethanol, air-dried, then extracted with water. The waterextract was lyophilized, resuspended in a small volume of water andsplit into three aliquots. Two aliquots were treated with glycolyticenzymes for 2 days at 37° C. (Flavobacterium heparin lyase III, 6.7mU/il, 50 mM sodium phosphate, pH 7.6, or Streptomyces HA lyase, 333milliunits/il, 50 mM sodium acetate, pH 5.8). The last aliquot wasmock-treated without enzyme in acetate buffer. The aliquots werequenched with SDS, subjected to paper chromatography, and the radiolabelat the origin was measured by liquid scintillation counting.

Results

Molecular Cloning of the Type D P. multocida Heparosan Synthase—A PCRproduct which contained a portion of the Type D UDP-glucosedehydrogenase gene was used as a hybridization probe to obtain the restof the Type D P. multocida capsular locus from a lambda library. Wefound a functional heparosan synthase, which we named pmHS, in severaldistinct Type D strains from different host organisms isolated aroundthe world. In every case, an open reading frame of 617 residues withvery similar amino acid sequence (98-99% identical) was obtained. In thelatter stages of our experiments, another group deposited a sequencefrom the capsular locus of a Type D organism in GenBank ¹⁵. In theirannotation, the carboxyl terminus of the pmHS homolog is truncated andmutated to form a 501-residue protein that was called DcbF (GenBankAccession Number AAK17905) (SEQ ID NO:17). No functional role for theprotein except “glycosyltransferase” was described and no activityexperiments were performed. As described herein, membranes or celllysates prepared from E. coli with the recombinant dcbF gene do notpossess heparosan synthase activity. The gene annotated as DcbF (SEQ IDNO:18) is truncated at the carboxyl terminus in comparison to thepresently claimed and described P. multocida HS clones. The truncated(T) or the full-length (FL) open reading frames of DcbF were cloned intothe expression system pETBlue-1 vector, as described hereinabove.Membranes isolated from the same host strain, E. coil Tuner with thevarious recombinant plasmids were tested in HS assays with bothradiolabeled UDP-sugars. The results of these experiments are summarizedin Table V.

TABLE V [14C]GlcUA Incorp. [3H]GlcNAc Incorp. Clone (dpm) (dpm) NegativeControl 160  40 B1(FL) 710(*) 1040(*) 012(T)  40  265 013(T)  70 1610019(T)  55 1105 N2(T)  70 1910 N4(T)  70  880 N5(T)  80  650

Five-fold less FL enzyme than T enzymes were tested in these parallelassays. At most, only a single GlcNAc sugar is added to the exogenouslysupplied acceptor in the truncated enzymes (T). Full-length HS from TypeD P. multocida, however, adds both sugars (*) to the nascent chain.Thus, the previously annotated and deposited DcbF gene is not afunctional heparosan synthase.

Another deduced gene was recently uncovered by the University ofMinnesota in their Type A P. multocida genome project ¹⁷, called PglA(GenBank Accession Number AAK02498), encoding 651 amino acids that aresimilar to pmHS (73% identical in the major overlapping region).However, the PglA gene is not located in the putative capsule locus.This group made no annotation of the function of PglA. Our studies showthat this PglA protein also polymerizes GlcUA and GlcNAc residues toform heparosan. We also found that a Type D strain and a Type F strainalso appear to contain a homologous PglA gene as shown by PCR andactivity analysis.

As mentioned before, during the pmHS cloning project in the presentinventor(s)' laboratory, investigators at the Univ. of Minnesotapublished the complete genome of a Pasteurella multocida isolate. Thefragments of the presently claimed and disclosed pmHS gene were utilizedas the query in a BLAST search. A gene annotated as pglA, but with noascribed, predicted or demonstrated function was found to be verysimilar to the pmHS gene. The pglA gene is not in the main capsule locusfound by either the DeAngelis or the Adler groups. The pglA open readingframe was obtained from two different encapsulated strains: Type A(P-1059 from a turkey—this strain is not the same as the Univ. ofMinnesota strain—clones denoted as “A”) and Type D (P-3881 from acow—clones denoted as “D”). The pglA gene was amplified from chromosomaltemplates prepared by method of Pitcher et al (Letters in AppliedMicrobiology, 1989). PCR with Taq polymerase (18 cycles) using customflanking oligonucleotide primers that correspond to the region of thestart codon and the stop codon of pglA. An appropriate size ampliconcorresponding to the pglA gene was found in both Type A and D strains;this result was rather unexpected if one considers that the capsularcompositions are HA and N-acetylheparosan polysaccharides, for Type Aand Type D strains, respectively. The resulting ˜1.9 kilobase PCRamplicons were ligated into an expression vector, pETBlue-1 (Novagen),transformed into the cloning host, E. coli Novablue (Novagen), andselected on LB carbenicillin and tetracycline plates at 30° C. Thecolonies were screened for the presence of insert in the properorientation by PCR with a combination of vector and insert primers.Clones were streak isolated, small cultures were grown, and preparationsof the plasmid DNA were made. The plasmids were transformed into theexpression host, E. coli Tuner (Novagen), and selected on LB withcarbenicillin and chloramphenicol.

After streak isolation, small cultures were grown at 30° C. as thestarting inoculum (1:100) for larger cultures (50 ml) for proteinexpression and activity assay. These cultures were grown in the same LBsupplemented with 1% casein amino acids and trace element solution withvigorous shaking (250 rpm) at 30° C. The cells were grown tomid-logarithmic phase (2.5 hours), induced with 0.5 mm IPTG, and grownfor 4.5 hours. Cells were collected by centrifugation and frozen at −80°C. overnight. The membrane preparations were isolated by coldlysozyme/ultrasonication method of DeAngelis et. al (J. Biol. Chem.,1998; pmHAS isolation the contents of which are expressly incorporatedherein in their entirety) except that 0.1 mM mercaptoethanol was used asthe reducing agent. The membranes were assayed for radioactive sugarincorporation and descending paper chromatography (according to themethodology of DeAngelis and Padget-McCue, J. Biol. Chem., 2000, thecontents of which are expressly incorporated herein in their entirety).

In general, a mixture with membranes, 50 mM Tris, pH 7.2, 10 mM MgCl₂,10 mM MnCl₂, 0.4 mM UDP-[³H]GlcNAc, 0.2 mM UDP-[¹⁴C]GlcUA, and heparinoligosaccharide acceptor (2 μg uronic acid) were incubated at 30° C. for2.5 hours before analysis by paper chromatography. As expected for apolysaccharide synthase, both sugars were incorporated into polymer(Table VI). Negative controls using membranes from a plasmid with anirrelevant control insert, do not show incorporation (data not shown).Therefore, PglA is a dual-action synthase capable of sugar biosynthesisas shown by functional expression of activity of one recombinant gene ina foreign host that normally does not make GlcUA/GlcNAc polymers. Therelaxed specificity of UDP-sugar incorporation of PglA should be of usefor the design and production of new polymers with alteredcharacteristics.

TABLE VI In vitro incorporation of sugar by membranes containingrecombinant pglA. CLONE [³H]GlcNAc (dpm) [¹⁴C]GlcUA (dpm) PglA-A2 50,40054,900 PglA-A4 39,100 41,000 PglA-D4 32,500 34,200 PglA-D7 44,800 46,600

The typical background for negative controls is less the 200 dpmincorporation. Type A and Type D isolates have the PglA, a synthase thatincorporates both GlcUA and GlcNAc sugars. (A=Type A; D=Type D;#=independent clone number).

Table VII shows PglA Sugar Specificity test results. The experimentssummarized in Table VII are similar to the experiments summarized inTable VI (with less enzyme) except that other UDP-sugars that are notnormally found in heparin or heparosan were also tested (note—60 minuteincubation times, 50 μl reactions). The Type A and the Type D enzymesbehave in a similar fashion with relaxed sugar specificity in this test.The PglA system can add a glucose instead of a GlcNAc sugar. The abilityto co-polymerize the sugars that compose the authentic heparin backbonewere tested by performing two parallel reactions:

-   UDP-[¹⁴C]GlcUA+various combinations of 2^(nd) UDP-sugars.-   UDP-[³H]GlcNAc+various combinations of 2^(nd) UDP-sugars.

TABLE VII Panel I. Type A PglA-A2 2^(nd) Sugar [³H]GlcNAc Incorporatedinto Polymer (dpm) none 450 UDP-GlcUA 12,900 UDP-GalUA 400 UDP-Glc 4302^(nd) Sugar [¹⁴C]GlcUA Incorporated into Polymer (dpm) none 60UDP-GlcNAc 7,700 UDP-GalNAc 60 UDP-Glc 985 Panel II. Type D PglA-D72^(nd) Sugar [³H]GlcNAc Incorporated into Polymer (dpm) none 570UDP-GlcUA 13,500 UDP-GalUA 530 UDP-Glc 500 2^(nd) Sugar [¹⁴C]GlcUAIncorporated into Polymer (dpm) none 60 UDP-GlcNAc 6,500 UDP-GalNAc 40UDP-Glc 660

TABLE VIII Acceptor Usage of PglA from Types A and D The Type A and theType D clones were tested for stimulation by addition of the Type Dpolysaccharide acceptor (described hereinbefore with respect to pmHS).Weaker stimulation of activity by acceptor on pglA was observed incomparison to pmHS (comparison is not shown here). [¹⁴C-GlcUA]incorporation Clone Acceptor NO Acceptor A2 1560 1210 D7 1240 1080

P. multocida Type F-derived recombinant pglA is thus also a heparosansynthase. As shown in the following Table IX, the Type F PglA canincorporate the authentic heparin sugars.

TABLE IX Activity of pglA from Type F ¹⁴C- ³H-GlcNAc GlcUA MembranesAcceptor (dpm) (dpm) Blank 0 8 8 PglA F 3 + 7100 3100 PglA F 4 0 61003800 PglA F 4 + 11000 6400 PglA F 18 0 20000 10000 PglA F 18 + 2300012000 PglA D 7 0 36000 17000

The pglA homolog of P. multocida Type F strain P-4218 was amplified withflanking primers as described for the Type A and D strains. The ORF wassubcloned into the pETBlue-1 system in E. coli Tuner cells for use as asource of membrane preparations as described. Three independent clones(F 3,4,18) were assayed under standard HS assay measuring radiolabeledsugar incorporation with paper chromatography. A negative control,membranes from “Blank” vector and a positive control, the Type D pglAclone D7, were tested in parallel. Reactions plus/minus the Type Dpolymer acceptor were assayed.

The next best heterologous matches for the pmHS enzyme in the Genbankdatabase are KfiA and KfiC proteins from E. coli K5; these two proteinswork together to make the heparosan polymer.^((7,8)) There is a goodoverall alignment of the enzyme sequences if smaller portions of pmHSORF are aligned separately with KfiA (pmHS2, SEQ ID NO:11)and KfiC(pmHS1, SEQ ID NO:10) (FIG. 1). The MULTALIN alignment program (Corpet,1988) identified regions that were very similar. Some of the mostnotable sequence similarities occur in the regions containing variantsof the DXD amino acid sequence motif. Indeed, the first 1-360 residuesof pmHS1 (denoted also as HSA1: SEQ ID NO:10) align with an approximate38% identity to the E. coli KfiC, a single action GlcUA-transferase,while the 361-617 residues of pmHS2 (denoted also HSA2: SEQ ID NO:11)align with an approximate 31% identity to the E. coli KfiA, aGlcNAc-transferase. Thus, the pmHS is a naturally occurring fusion oftwo different glycosyltransferase domains. The pmHS is a dual actionenzyme that alone makes heparin/heparosan polymers because both sugartransferase sites exist in one polypeptide enzyme.

Heterologous Expression of a Functional P. multocida Heparosan Synthase—

Membrane extracts derived from E. coli Tuner cells containing theplasmid encoding pmHS, but not samples from cells with the vector alone,synthesized polymer in vitro when supplied with both UDP-GlcUA andUDP-GlcNAc simultaneously. The identity of the polymer as heparosan wasverified by its sensitivity to Flavobacterium heparin lyase III (˜97%destroyed after treatment) and its resistance to the action ofStreptomyces HA lyase. No substantial incorporation of radiolabeled[¹⁴C]GlcUA into polymer was observed if UDP-GlcNAc was omitted, or ifUDP-GalNAc was substituted for UDP-GlcNAc. Conversely, in experimentsusing UDP-[³H]GlcNAc, substantial incorporation of radiolabel intopolymer was only noted when UDP-GlcUA was also present. UDP-GalUA orUDP-Glc did not substitute for UDP-GlcUA. No polymerization ortransferase activity was detected if the divalent metal ions werechelated with EDTA. Maximal activity was observed in reactions thatcontained Mn ion, but Mg also supported substantial incorporation(65%-85% maximal). Cobalt was a poorer cofactor (˜30% maximal). Theaddition of the heparosan polymer acceptor increased sugar incorporationcatalyzed by pmHS at least 7- to 25-fold in comparison to parallelreactions without acceptor (FIG. 2) in analogy to observations ofpmHAS¹⁸ and pmCS¹⁴. The acceptor stimulation of activity is due to thelower efficiency or slower rate of initiation of a new polymer chain incomparison to the elongation stage in vitro. The exogenous acceptorsugar associates with the recombinant enzyme's binding site for thenascent chain and then is elongated rapidly.

Analysis by gel filtration chromatography indicated that recombinantpmHS produced long polymer chains (˜1-3×103 monosaccharides or ˜200-600kDa) in vitro without acceptor (FIG. 3). If acceptor polymer wassupplied to parallel reaction mixtures, then higher levels of shorterchains (˜0.1-2×103 monosaccharides or ˜20-400 kDa added to acceptor)were more rapidly produced. Radioactivity from both labeled GlcUA andGlcNAc sugars co-migrated as a single peak in all chromatographyprofiles. Some chains also appear to be initiated de novo in reactionswith acceptor as evidenced by the small peak of higher molecular weightmaterial near the void volume. Apparently, once pmHS either starts a newchain or binds an existing chain, then rapid elongation is performed.

We found in parallel tests that membranes or lysates prepared fromrecombinant cells containing the predicted dbcF gene⁵ (SEQ ID NO:17), atruncated version of pmHS, in the same expression vector, do not exhibitheparosan synthase activity. Even with large amounts of total protein,repeated polymerization was not observed and no significant radiolabelincorporation above background levels was noted.

Discussion

We have molecularly cloned a dual-action glycosyltransferase responsiblefor polymerizing the heparosan backbone component of the Type D P.multocida capsular polysaccharide. As discussed earlier, the first 497residues of the pmHS protein are virtually identical to the hypotheticalDcbF sequence. We have sequenced the DNA from the equivalent P-934isolate obtained from the same USDA collection as reported¹⁵, as well asseveral other Type D strains, but our results do not agree with the dcbFopen reading frame. The Adler group appears to have made a sequencingerror that resulted in a frame-shift mutation; a conceptual prematuretermination led to the creation of the erroneously truncated dcbFannotation. Recently, we have determined that the Pasteurella hyaluronansynthase, pmHAS, contains two active sites in a single polypeptide bygenerating mutants that transfer only GlcUA or only GlcNAc¹⁹.Interestingly, mixing the two different mutant pmHAS proteinsreconstituted the HA synthase activity. We hypothesized that one domain,called A1, is responsible for GlcNAc transfer and the other domain,called A2, is responsible for GlcUA transfer¹⁹. The chondroitinsynthase, pmCS, transfers a different hexosamine, GalNAc, and alsoappears to contain a similar two-domain structure¹⁴. The amino acidsequence of the heparosan synthase, pmHS, however, is very differentfrom other Pasteurella GAG synthases, pmHAS and pmCS. The pmHAS and pmHSenzymes both perform the task of polymerizing the identicalmonosaccharides; HA and heparin only differ with respect to theirlinkages. The creation of different anomeric linkages probably requiresvery distinct active sites due to the disparity between a retaining (toform α-linkages) and an inverting (to form β-linkages) transfermechanism. The putative dual-action vertebrate heparin synthases, EXT1(SEQ ID NO:19) and EXT 2 (SEQ ID NO:20), also appears to have twotransferase domains, but the amino acid sequences are not similar topmHS²⁰. Thus, by aligning pglA, pmHS (B10 and A2 clones), KfiA, or KfiC,deduced amino acid sequence motifs have been identified. Such motifs arelisted below and the alignment is shown in FIGS. 4A-G.

Two distinct regions of pmHS (pmHS1 and pmHS2) are similar to the E.coli K5 KfiA or KfiC proteins suggesting the limits of the sugartransfer domains (FIG. 1). On the basis of sequence similarity, if theKfi studies are correct, then GlcUA transfer and GlcNAc transfer occurat the amino and carboxyl portions of pmHS, respectively. The pmHSprotein may be the result of the fusion of two ancestral single-actionenzymes. The efficiency and convenience of combining the two requiredenzyme activities into a single polypeptide seems clear, but as acounterexample, the E. coli KfiA and KfiC proteins remain separateentities. Interestingly, pglA, a gene with no reported function from aType A¹⁷ isolate is (˜70%) similar to the pmHS gene of a Type D strain.In parallel expression experiments, PglA from Type A or D or F strainsalso appear to be heparosan synthases, as shown hereinabove. It is quitepuzzling that the Type A strain would have a heparosan synthase as wellas the known HA synthase. The major Type A capsular polymer was shown tobe HA, but in retrospect, a small amount of heparosan would be difficultor impossible to detect in these characterization studies¹¹. A possiblescenario for the presence of a heparosan synthase in the Type A bacteriais that the pglA gene is repressed or silent and not expressed in thishost under laboratory conditions. The pglA gene could also be a crypticremnant from an ancestral organism (i.e. before Types A and D and Fdiverged) that has been maintained and the gene product is stillfunctional. Another interesting possibility is that in Type A organismseither the pmHAS or the pglA gene is utilized at different timesdepending on conditions or stage of infection; using different capsularpolymers could serve as a phase-shift mechanism.

Comparisons of the two known sets of heparin/heparosan biosynthesisenzymes from the E. coli K5 Kfi locus, the PglA enzyme, and the pmHSfrom Type D capsular locus, allows for the initial assessment andbioinformatic prediction of new enzymes based on the amino acid sequencedata. The closer the match (% identity) in a single polypeptide for thetwo sequence motifs described hereinafter (corresponding to the criticalelements of the GlcUA-transferase and the GlcNAc-transferase), thehigher the probability that the query enzyme is a new heparin/heparosansynthase (a single dual-action enzyme). The closer the match (%identity) in two polypeptides (especially if encoded in the same operonor transcriptional unit) for the two sequence motifs, the higher theprobability that the query enzymes are a pair of single-actionglycosyltransferases. Thus, one of ordinary skill in the art wouldappreciate that given the following motifs, one would be able toascertain and ascribe a probable heparin synthase function to a newlydiscovered enzyme and then test this ascribed function in a manner toconfirm the enzymatic activity. Thus, single dual-action enzymespossessing enzymatic activity to produce heparin/heparosan and having atleast one of the two disclosed motifs are contemplated as beingencompassed by the presently claimed and disclosed invention.

Motif I: (SEQ ID NO:21)

-   QTYXN(L/I)EX₄DDX(S/T)(S/T)D(K/N)(T/S)X₆IAX(S/T)(S/T)(S/T)(K/R)V(K/R)X₆NXGXYX₁₆FQDXDDX(C/S)H(H/P)ERIXR    Motif II: (SEQ ID NO:22)-   (K/R)DXGKFIX₁₂₋₁₇DDDI(R/I)YPXDYX₃MX₄₀-50VNXLGTGTV

Motif I corresponds to the GlcUA transferase portion of the enzyme,while Motif II corresponds to the GlcNAc transferase portion of theenzyme. With respect to the motifs:

-   X=any residue-   parentheses enclose a subset of potential residues [separated by a    slash] that may be at a particular position (e.g.—(K/R) indicates    that either K or R may be found at the position—i.e. there are    semiconserved residues at that position.

The consensus X spacing is shown with the number of residues insubscript (e.g. X₁₂₋₁₇), but there are weaker constraints on theseparticular residues, thus spacing may be longer or shorter. Conservedresidues may be slightly different in a few places especially if achemically similar amino acid is substituted (e.g. K for a R, or E for aD). Overall, at the 90% match level, the confidence in this predictivemethod is very high, but even a 70-50% match level without excessive gapintroduction (e.g. altered spacing between conserved residues) orrearrangements (miss-positioning with respect to order of appearance inthe amino to carboxyl direction) would also be considered to be withinthe scope of these motifs. One of ordinary skill in the art, given thepresent specification, general knowledge of the art, as well as theextensive literature of sequence similarity and sequence statistics(e.g. the BLAST information website at www.ncbi.nlm.mih.gov), wouldappreciate the ability of a practitioner to identify potential newheparin/heparosan synthases based upon sequence similarity or adherenceto the motifs presented herein and thereafter test for functionality bymeans of heterozologous expression, to name but one example.

Bacteria-derived heparosan may be converted by epimerization andsulfation into a polymer that resembles the mammalian heparin andheparan sulfate because all the modifying enzymes have been identified³.In general, sulfation with chemical reagents (SO₃, chlorosulfonic acid)or sulfo-transferases (i.e. 2-0-GlcUA-sulfotransferase, etc.) and PAPsprecursor is possible. N-sulfation can be done by using either chemicalmeans (hydrazinolysis and subsequent N-sulfation) or enzymatic meanswith dual function deacetylase/N-sulfotransferase. For creation ofiduronic acid, epimerization can be performed enzymatically with heparinepimerase or chemically with super-critical carbon dioxide. The art isreplete with articles, methods, and procedures for sulfating andepimerizing heparosan to form heparin. Example, include Leali, et al.,Fibroblast Growth Factor-2 Antagonist Activity and Angiostatic Capacityof Sulfated E.coli KS Polysaccharide Derivatives, J. Biol. Chem., Vol.276, No. 41, Oct. 12, 2001, pp. 37900-902; Esko, et al., MolecularDiversity of Heparin Sulfate, J. Clin. Invest. 108: 169-173 (2001); andCrawford, et al., Cloning, Golgi Localization, and Enzyme Activity ofthe Full-Length Heparin/Heparosan Sulfate—Glucuronic Acid C5-Epimerase,J. Biol. Chem., Vol. 276, No. 24, Jun. 15, 2001, pp. 21530-543, thecontents of each being hereby expressly incorporated by reference intheir entirety. Thus, given the present specification which disclosesand teaches methods for the recombinant production of Heparosan, one ofordinary skill in the art would be capable of producing Heparintherefrom. As such, Heparin obtained through the process of sulfatingand epimerizing Heparosan is contemplated as falling within the scope ofthe presently disclosed and claimed invention.

Type D P. multocida with pmHS or PglA (or an improved recombinantversion) may be a more economical and useful source of heparosan than E.coil K5 for several reasons. Pasteurella has a higher intrinsicbiosynthetic capacity for capsule production. The Pasteurella capsuleradius often exceeds the cell diameter when observed by light microscopyof India Ink-prepared cells. On the other hand, visualization of themeager E. coil K5 capsule often requires electron microscopy. From asafety standpoint, E. coil K5 is a human pathogen, while Type DPasteurella has only been reported to cause disease in animals.Furthermore, with respect to recombinant gene manipulation to createbetter production hosts, the benefits of handling only a single geneencoding pmHS or PglA, dual action synthases, in comparison to utilizingKfiA and C (and probably KfiB) are obvious. The in vitro properties ofpmHS and pglA are also superior; these enzymes can make large chains invitro either with or without an exogenous acceptor sugar, but KfiA andKfiC do not.

The discovery of pmHS and PglA expands the known GAG biosynthesisrepertoire of P. multocida. Depending on the Carter capsular type, thiswidespread species produces HA, heparosan, or chondroitin.

Thus, it should be apparent that there has been provided in accordancewith the present invention purified nucleic acid segments having codingregions encoding enzymatically active heparosan synthase, methods ofproducing heparosan from the pmHS or pglA gene, and the use of heparosanproduced from a heparosan synthase encoded by the pmHS or pglA gene,that fully satisfies the objectives and advantages set forth above.Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives,modifications, and variations will be apparent to those skilled in theart. Accordingly, it is intended to embrace all such alternatives,modifications, and variations that fall within the spirit and broadscope of the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   1. Roden, L. (1980) in The Biochemistry of Glycoproteins and    Proteoglycans (Lennarz, W. J., ed) pp. 267-371, Plenum Publishing    Corp., New York-   2. Lidholt, K. (1997) Biochem. Soc. Trans. 25, 866-870-   3. Esko, J. D. and Lindahl U. (2001) J. Clin. Invest., 108:169-173-   4. Roberts, I. S. (1996) Annu. Rev. Microbiol. 50, 285-315-   5. Vann, W. F., Schmidt, M. A., Jann, B., and Jann, K. (1981)    Eur. J. Biochem. 116, 359-364-   6. Rodriguez, M. L., Jann, B., and Jann, K. (1988) Eur. J. Biochem.    177, 117-124-   7. Griffiths, G., Cook, N. J., Gottfridson, E., Lind, T., Lidholt,    K., and Roberts, I. S. (1998) J. Biol. Chem., 273,11752-11757-   8. Hodson, N., Griffiths, G., Cook, N., Pourhossein, M.,    Gottfridson, E., Lind, T., Lidholt, K. and Roberts, I. S. (2000) J.    Biol. Chem., 275:27311-27315.-   9. Rimler, R. B. (1994) Vet. Rec. 134, 191-192-   10. Rimler, R. B., Register, K. B., Magyar, T., and    Ackermann, M. R. (1995) Vet. Microbiol. 47, 287-294-   11. Rosner, H., Grimmecke, H. D., Knirel, Y. A., and    Shashkov, A. S. (1992) Carb. Res. 223, 329-333-   12. DeAngelis, P. L., Jing, W., Drake, R. R. and    Achyuthan, A. M. (1998) J. Biol. Chem. 273, 8454-8458-   13. Rimler, R. B. and Rhoades, K. R. (1987) J. Clinic. Microbiol.    25, 615-618-   14. DeAngelis, P. L. and Padgett-McCue, A. J. (2000) J. Biol. Chem.,    275: 24124-24129-   15. Townsend, K. M., Boyce, J. D., Chung, J. Y., Frost, A. J., and    Adler, B. (2001) J. Clin. Microbiol. 39:924-929-   16. DeAngelis, P. L., and Weigel, P. H. (1994) Biochem. 33,    9033-9039-   17. May, B. J., Zhang, Q., Li, L. L., Paustian, M. L., Whittam, T.    S., and Kapur, V. (2001) Proc. Natl. Acad. Sci. U.S.A. 98:3460-3465-   18. DeAngelis, P. L. (1999) J. Biol. Chem. 274, 26557-26562-   19. Jing, W. and DeAngelis, P. L. (2000) Glycobiology 10: 883-889-   20. Duncan, G., McCormick, C., and Tufaro, F. (2001) J. Clin.    Invest.108: 511-516-   21. Corpet, F. (1988) Nucleic Acids Res. 16:10881-10890 18

1. A purified nucleic acid segment having a coding region encodingheparin synthase, wherein the heparin synthase is a single protein thatis a dual-action catalyst that utilizes UDP-GlcUA and UDP-GlcNAc tosynthesize heparin, wherein the purified nucleic acid segment is atruncated segment when compared to the nucleotide sequence of SEQ IDNO:1 or
 3. 2. A purified nucleic acid segment comprising a coding regionencoding heparin synthase, wherein the heparin synthase is a singleprotein that is a dual-action catalyst that utilizes UDP-GlcUA andUDP-GlcNAc to synthesize heparin, and wherein the purified nucleic acidsegment encodes the Pasteurella multocida heparin synthase of SEQ IDNO:2 or
 4. 3. A purified nucleic acid segment comprising a coding regionencoding heparin synthase, wherein the heparin synthase is a singleprotein that is a dual-action catalyst that utilizes UDP-GlcUA andUDP-GlcNAc to synthesize heparin, and wherein the purified nucleic acidsegment comprises a nucleotide sequence in accordance with SEQ ID NO:1or
 3. 4. A recombinant vector selected from the group consisting of aplasmid, cosmid, phage, integrated cassette or virus vector and whereinthe recombinant vector further comprises a purified nucleic acid segmenthaving a coding region encoding heparin synthase, wherein the heparinsynthase is a single protein that is a dual-action catalyst thatutilizes UDP-GlcUA and UDP-GlcNAc to synthesize heparin, and wherein thepurified nucleic acid segment encodes the Pasteurella multocida heparinsynthase of SEQ ID NO:2 or
 4. 5. The recombinant vector of claim 4,wherein the plasmid further comprises an expression vector.
 6. Therecombinant vector of claim 4, wherein the expression vector comprises apromoter operatively linked to the heparin synthase coding region.
 7. Arecombinant vector selected from the group consisting of a plasmid,cosmid, phage, integrated cassette or virus vector and wherein therecombinant vector further comprises a purified nucleic acid segmenthaving a coding region encoding heparin synthase, wherein the heparinsynthase is a single protein that is a dual-action catalyst thatutilizes UDP-GlcUA and UDP-GlcNAc to synthesize heparin, and wherein thepurified nucleic acid segment comprises a nucleotide sequence inaccordance with SEQ ID NO:1 or
 3. 8. The recombinant vector of claim 7,wherein the plasmid further comprises an expression vector.
 9. Therecombinant vector of claim 7, wherein the expression vector comprises apromoter operatively linked to the heparin synthase coding region.
 10. Arecombinant host cell, wherein the recombinant host cell iselectroporated, transformed, transfected or transduced to introduce arecombinant vector into the recombinant host cell, wherein therecombinant vector comprises a purified nucleic acid segment having acoding region encoding heparin synthase, wherein the heparin synthase isa single protein that is a dual-action catalyst that utilizes UDP-GlcUAand UDP-GlcNAc to synthesize heparin, and wherein the purified nucleicacid segment encodes the Pasteurella multocida heparin synthase of SEQID NO:2 or
 4. 11. The recombinant host cell of claim 10, wherein thehost cell produces heparin.
 12. The recombinant host cell of claim 10,wherein the recombinant host cell is a prokaryotic cell.
 13. Therecombinant host cell of claim 10, wherein the recombinant host cell isa eukaryotic cell.
 14. A recombinant host cell, wherein the recombinanthost cell is electroporated, transformed, transfected or transduced tointroduce a recombinant vector into the recombinant host cell, whereinthe recombinant vector comprises a purified nucleic acid segment havinga coding region encoding heparin synthase, wherein the heparin synthaseis a single protein that is a dual-action catalyst that utilizesUDP-GlcUA and UDP-GlcNAc to synthesize heparin, and wherein the purifiednucleic acid segment comprises a nucleotide sequence in accordance withSEQ ID NO:1 or
 3. 15. The recombinant host cell of claim 14, wherein thehost cell produces heparin.
 16. The recombinant host cell of claim 14,wherein the recombinant host cell is a prokaryotic cell.
 17. Therecombinant host cell of claim 14, wherein the recombinant host cell isa eukaryotic cell.